Ras proteins are evolutionarily conserved, membrane-associated, small GTP binding proteins involved in signal transduction pathways. Given the high incidence of Ras mutations in human cancers, considerable effort has gone into studying the function of Ras proteins and ways to inhibit Ras activity. One particularly promising approach has been to target the enzymes required for the posttranslational addtion of farnesyl and palmitoyl lipids. Farnesylation and the farnesyl transferase (FT) enzymes have been extensively studied, but much less is known about the palmitoylation of Ras or other palmitoylated proteins. The covalent attachment of palmitate to eukaryotic proteins was first described over 30 years ago and since that time the list of acylated proteins has grown. The types of proteins that undergo palmitoylation are quite diverse and include intrinsic and peripherally associated membrane proteins. Palmitoylation is important in receptor trafficking and recycling, protein association with lipid microdomains, vesicle fusion, and signal transduction. While the identification of palmitoylated proteins has proceeded rapidly, understanding of the molecular mechanisms that underlie modification with palmitate has advanced more slowly. The reversibility of palmitoylation raises the possibility that it is a regulatory modification much like protein phosphorylation. During the last funding period, we identified the Ras palmitoyltransferase (PAT). The discovery of the Ras PAT filled a long-standing gap in the field of protein palmitoylation. The current proposal is divided into five specific aims that address basic properties of Ras PAT enzymes including membrane topology, the role of zinc in structure and catalysis, subunit interactions, substrate specificity, and regulation by phosphorylation. This study will provide essential new information about a class of lipid modification enzymes with potential impact to human health. The goals of this study fall within the general area of "Molecular Targets of Prevention, Diagnosis, and Treatment", one of the five priority areas identified in the 2005 National Investment in Cancer Research (NCI).